Elucidation of eukaryotic membrane protein structures is limited in comparison to non-membrane associated proteins in part due to the inability to express sufficient amounts of functional proteins in heterologous hosts. Mammalian membrane proteins (MMPs) are particularly difficult to overexpress in functional conformations useful for structural determination due to their complex folding and processing patterns and tendency to accumulate in unfolded and non-functional forms. For example, the four biomedically important MMPs: serotonin transporter (SERT), neurotensin receptor (NTR), mesenchymal-epithelial transition factor (MET) receptor, and voltage-sensitive sodium channel (Nav1.7) all have complex post-translational processing requirements. Optimal expression of these MMPs often occurs when the protein is expressed in a host that is evolutionarily closest to the target MMP. Indeed, NTR and SERT are expressed at their highest functional levels on a per cell basis in mammalian hosts. However, mammalian and other vertebrate cell lines have been used sparingly due to their low overall MMP yields of 0.05 mg/L or lower. If the yields of MMPs could be increased by an order of magnitude or more, mammalian (and other vertebrate) culture systems would be used more widely due to their superior capabilities for processing complex MMP structural targets into a functional form. Culture requirements would be reduced from hundreds to tens of liters. A hypothesis of this proposal is that the nature of mammalian membrane protein sequence and the processing efficiency of the current hosts makes it problematic to express functional proteins at high levels in heterologous hosts. The very low natural abundance of some complex MMPs has minimized the natural evolutionary expression pressures and limited the need to minimize their toxicity on hosts. Therefore, the goal of the current proposed project is to develop technologies to improve functional expression levels by the following two aims: 1) evolving protein structure to identify those amino residues whose mutagenesis can improve expression without altering structure or function significantly 2) engineering and evolving mammalian hosts through mutagenesis to generate cell lines able to process complex MMPs more efficiently. In situ evolution and selection technologies will be implemented that allow users to mutate the sequence of target MMPs in a somatic hypermutating B cell host. In parallel, mammalian production hosts will be engineered with anti-apoptotic proteins, molecular chaperones, and transcription factors capable of improving protein processing. Furthermore, directed evolution of cell lines will be undertaken by inhibiting the cells natural DNA repair machinery. This approach will be tested on the four MMP targets above that represent different classes of MMPs and are related to a number of diseases. The mutated proteins variants will be expressed in engineered and evolved mammalian cell lines in order to increase active product yields by 10 to 20 fold for ongoing crystallography projects. This project will provide a new paradigm for expressing high yields of complex MMPs based on protein and cellular evolution strategies.